Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport

Detailed investigations of proximal tubular function in Imerslund-Grasbeck syndrome

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B(12) receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B(12) binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria

Multiscale molecular dynamics simulations of human P-glycoprotein in complex lipid bilayer

The topology (gro) and trajectory (xtc) files for multiscale - coarse-grained (CG) and atomistic (AT) molecular dynamics simulations of human P-glycoprotein in complex lipid bilayer. The coarse grained simulations are 10 microseconds long and the trajectories have 1 frame saved at every 10 ns, while the atomistic simulations are 100 ns long and have 2 frames saved at every nanosecond

Local Lipid Reorganization by a Transmembrane Protein Domain

Membrane proteins interact with their lipid bilayer environment via both a transmembrane helix and juxtamembrane regions. The effect of juxtamembrane regions and membrane lipid composition on these interactions has been explored by multiscale molecular dynamics simulations. The consequences of anionic lipids within the inner leaflet of a membrane were studied in combination with membrane spanning protein models differing in their juxtamembrane domains. The simulations reveal sensitivity of the protein–lipid interactions to membrane lipid composition and charged amino acid side chains. Basic residues on the intracellular side of the protein facilitated interactions with anionic lipids. Protein systems without basic residues do not show selectivity for anionic compared with zwitterionic lipids. This reveals the sensitivity to the composition of both the membrane and the protein system when studying membrane-embedded proteins. The results presented here illustrate how even a simple transmembrane domain is able to induce lipid reorganization in a mixed asymmetric bilayer